CN110286189A - Nephrotic syndrome lesion process associated metabolic marker and its application - Google Patents
Nephrotic syndrome lesion process associated metabolic marker and its application Download PDFInfo
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Abstract
The invention belongs to nephrotic syndrome marker screening and identification technical field, a kind of nephrotic syndrome lesion process associated metabolic marker and its application are provided.Bis- (4- Ethylbenzyl) sorbierites, C16 dihydrosphingosine, lysophosphatidyl ethanolamine (22:6), lysophosphatidyl choline (22:5), N- ethanol amine, proline are progressivity marker；Oleamide, docosahexaenoic acid, 14S- hydroxyl-docosahexaenoic acid, clupanodonic acid, uric acid, lysophosphatidyl choline (20:5) and (18:1) are early sign object；Acylcarnitines C18:0; linoleic acid carnitine; lysophosphatidyl choline (18:3), DL-tryptophan, indole acrylic acid; paracresol sulfate; 3- indoxylsulfuric acid salt, cytimidine, kreatinin; sphingosine-1-phosphate, L- Su Shi-dihydrosphingosine and ceramide (d18:0/14:0) are late-stage markers object.It can quantify, rapid sensitive degree height, high specificity.
Description
Technical field
The invention belongs to nephrotic syndrome marker screening and identification technical fields, and in particular to nephrotic syndrome lesion process
Associated metabolic marker and its application.Specifically, using two kinds of Adriamycin-induced Nephropathies and LC-MS technology analysis obtain with
The relevant marker of nephrotic syndrome lesion process and its use in preparation nephrotic syndrome lesion process diagnosis different reagent
On the way.
Background technique
Nephrotic syndrome shows High-grade Proteinuria, and the symptom of Hypoproteinemia and hyperlipidemia is due to the various causes of disease
The syndrome that caused glomerular filtration membrane permeability changes, and nephrotic syndrome is the multiple intractable disease of children
Disease, disease incidence is high, seriously affects human health.Clinical diagnosis is mainly horizontal according to quantity of proteinuria and plasma albumin；Tool
Body pathology is judged according to needs according to renal biopsy tissue pathology.The former specificity of diagnosis basis is poor, the latter's patient dependence
Difference, the treatment and prognosis to the disease are all very unfavorable, thus it is extremely important to establish non-intrusive fast diagnosis method, especially searching energy
Enough potential pathological hallmark objects as reflection nephrotic syndrome lesion process, the deterioration to renal function is slowed down, prevention complication are outstanding
For key.
The Nephrotic Syndrome in Rats of adriamycin induction is the animal model of generally acknowledged simulation human renal diseases, clinical manifestation
Close to the feature of nephrotic syndrome.Can judge according to 24 h quantity of proteinuria in experiment nephrotic syndrome model copy success with
It is no, and the histological type of nephrosis and lesion degree are mainly checked according to renal pathology, not only subjectivity is strong and takes time and effort,
It there is no nephrotic syndrome lesion process dynamic evaluation method at present, thus it is non-intrusive, quick, highly sensitive to need to establish a kind of entirety
The method of the good course of disease that can be used for diagnosis of nephropathy and dynamically reflect disease of degree, specificity.
Metabolism group is answered in many fields with the high throughput ability of its ever-increasing coverage area and its inherence
With such as medical diagnosis on disease and treatment, drug toxicity research, biomarker discovery and disease mechanisms exploration etc..Thus, using Ah
Mycin Nephrotic Syndrome in Rats simulates nephrotic syndrome, comprehensive with nephrosis in conjunction with LC-M metabonomic technology and mathematical statistics method searching
The relevant potential marker of simulator sickness lesion process, to find potential pathological hallmark relevant to nephrotic syndrome Development process
Object provides Research foundation for disease diagnosis, treatment and prognosis.
Summary of the invention
The present invention can determine that nephrotic syndrome lesion process to solve the disadvantage that the prior art not, provide a kind of nephrosis
Syndrome lesion process associated metabolic marker and its application.
The present invention is realized by following technical solution: a kind of nephrotic syndrome lesion process associated metabolic marker, described
Metabolic markers are bis- (4- Ethylbenzyl) sorbierites, C16 dihydrosphingosine, lysophosphatidyl ethanolamine (22:6), haemolysis phosphorus
Phosphatidylcholine (22:5), N- ethanol amine, proline is as progressivity marker；Oleamide, docosahexaenoic acid, 14S- hydroxyl
Base-docosahexaenoic acid, clupanodonic acid, uric acid, lysophosphatidyl choline (20:5) and lysophosphatidyl choline
(18:1) is used as early sign object；Acylcarnitines C18:0, linoleic acid carnitine, lysophosphatidyl choline (18:3), DL- color
Propylhomoserin, indole acrylic acid, paracresol sulfate, 3- indoxylsulfuric acid salt, cytimidine, kreatinin, sphingosine-1-phosphate, L- Su Shi-
Dihydrosphingosine and ceramide (d18:0/14:0) are used as late-stage markers object.
In all metabolic markers, by taking ceramide (d18:0/14:0) as an example, d indicates that there are two hydroxyl, N in structural formula
Atom connects two Long carbon chains, and one has above 18 carbon and has 0 double bond (i.e. 18:1)；Another has 12 carbon, does not have above
Double bond (i.e. 14:0).
A kind of method of screening nephrotic syndrome lesion process associated metabolic marker, steps are as follows:
The duplication of (1) two kind of Adriamycin-induced Nephropathy and tradition are evaluated: rat is randomly divided into blank control group, early stage adriamycin
Nephropathy model group and advanced stage Adriamycin-induced Nephropathy group, early stage Adriamycin-induced Nephropathy group and advanced stage Adriamycin-induced Nephropathy group are adopted
It is induced with tail vein injection adriamycin, establishes Adriamycin-induced Nephropathy；Utilize renal pathology degree of injury and Urine proteins
Quantitative level evaluates Adriamycin-induced Nephropathy；
(2) searching of difference metabolin: collecting the blood serum sample of each group rat, carries out LC-MS analysis to serum, belongs to and be metabolized out
Object；Liquid matter map is converted to data matrix through processing, imported into SIMCA-PSoftware carries out the searching of difference metabolin；
(3) marker for combining statistical method screening, determining reflection nephrotic syndrome lesion process；
(4) based on the relative amount of metabolin, metabolin is confirmed using thermal map, correlation analysis figure, and uses ROC curve
The diagnosis capability of marker is assessed, early stage, advanced stage and the progressivity mark of reaction nephrotic syndrome model are finally determined
Will object.
The duplication of two kinds of Adriamycin-induced Nephropathies in step (1) method particularly includes: rat adapts to environment one week, collects big
The urine of 24 h of mouse, and record volume of urine；It is according to progress primary dcreening operation, by qualified rat with the quantity of proteinuria of rat and weight
It is randomly divided into blank control group and early stage Adriamycin-induced Nephropathy group and advanced stage Adriamycin-induced Nephropathy group, freedom in experimentation
Drinking-water feed；Early stage Adriamycin-induced Nephropathy group rat is in the adriamycin of the 1st day 4 mg/kg of tail vein injection, then every 1 week,
In the adriamycin of the 8th day 2 mg/kg of tail vein injection, mRNA IN ADRIAMYCIN NEPHROPATHY early stage model is replicated；Advanced stage Adriamycin-induced Nephropathy group
Rat replicates mRNA IN ADRIAMYCIN NEPHROPATHY advanced stage model in the adriamycin of the 1st day 6 mg/kg of disposable tail vein injection；Blank control group
Isodose physiological saline is given in an identical manner；
During model copy, respectively at the 0th day, the 7th day, the 14th day, the 28th day, 9:00 in the 42nd day morning by blank control
Group and early and late Adriamycin-induced Nephropathy group rat are respectively placed in rat metabolism cage, and Yu Ci 9:00 in morning collects urine
Liquid, and volume of urine is recorded, supernatant is taken after 4 DEG C, 3500 rpm centrifugation, 15 min to the urine sample of collection, is placed in -80
DEG C refrigerator is spare；
Quantity of proteinuria analysis is carried out using double pungent butyric acid BCA methods to urine, 24 h urine of rat is quantitative determined at 562 nm
Absorbance, establishing criteria curve calculate urine protein concentration, are counted by the way that urinary protein concentrations mg/mL is urinated volume mL multiplied by 24 h
Calculate 24 h excretion quantity of urinary protein Upro.
Blood serum sample carries out LC-MS analysis method particularly includes: uses HESI Ionization mode, spray voltage: anode,
3.5kV；Cathode, 2.5kV；320 DEG C of capillary temperature；300 DEG C of heter temperature；Sheath gas: 35arb, secondary air speed:
10arb；Scan pattern is Full Scan/dd-MS2, acquisition range m/z100-1500, cation switching acquisition mode；Point
Resolution uses MS Full Scan 35000 FWHM, MS/MS17500FWHM, NCE 12.5eV, 25eV and 37.5eV；
All original documents, including blank control group and early and late Adriamycin-induced Nephropathy group, are all to pass through Xcalibur
Derived from work station and Compound Discover 2.0, an independent peak value meter m/z and retention time have been obtained；It is all
Sample is all made of following parameter and is studied: S/N threshold value: 3, mass range: 100 ~ 1500 Da, quality tolerance: 5 ppm.
Blood serum sample data processing method in step (2) are as follows: data matrix is imported into SIMCA-PIn 13.0 softwares, with sky
White control group and early and late Adriamycin-induced Nephropathy group construct unsupervised pattern-recognition principal component analysis PCA, then structure
Partial Least Squares discriminant analysis PLS-DA is built, arranges the reliability of experimental evaluation model, and for subsequent difference metabolin
Identification；Constructing coefficient > 0.7 V+S-plot combines VIP > 1 to find to the biggish difference metabolin of the two grouping contribution,
Pass throught- test(p< 0.05) the difference metabolin with conspicuousness is determined.
Step (3) method particularly includes: according to S-plot and VIP value, select the mass ions with statistically-significant difference
VIP>1.0 and P<0.05 are determined as the difference object marker of identification；The difference metabolin that early and late model has is thought
It is progressivity marker；
According to S-plot and VIP value, blank control group and early stage Adriamycin-induced Nephropathy group are analyzed, selecting has statistics
Mass ions VIP>1.0 and P<0.05 for learning significant difference, are determined as potential early sign object；With above-mentioned potential early sign object
It is variable with rat quantity of proteinuria, by differentiating the data characteristics of every group of data, selects correlation analysis method, calculate the two
Related coefficient determines early sign object；According to area under ROC curve, using the metabolin of AUC > 0.7 as reflection nephrotic syndrome
Early sign object；
According to S-plot and VIP value, blank control group and advanced stage Adriamycin-induced Nephropathy group are analyzed, selecting has statistics
Mass ions VIP>1.0 and P<0.05 for learning significant difference, are determined as potential late-stage markers object；With above-mentioned potential late-stage markers object
It is variable with rat quantity of proteinuria, by differentiating the data characteristics of every group of data, selects correlation analysis method, calculate the two
Related coefficient determines late-stage markers object；According to area under ROC curve, using the metabolin of AUC > 0.7 as reflection nephrotic syndrome
Late-stage markers object.
It is prepared by a kind of application of nephrotic syndrome lesion process associated metabolic marker, the metabolic markers
Purposes in nephrotic syndrome lesion process diagnosis different reagent.
The present invention has finally determined the marker of reflection nephrotic syndrome progressive disease.I.e. with bis- (4- Ethylbenzyl) mountains
Pears alcohol, C16 dihydrosphingosine, lysophosphatidyl ethanolamine (22:6), lysophosphatidyl choline (22:5), N- ethanol amine, dried meat ammonia
Acid is used as progressivity marker；Oleamide, docosahexaenoic acid, 14S- hydroxyl-docosahexaenoic acid, 22 light dydrocarbons
Olefin(e) acid, uric acid, lysophosphatidyl choline (20:5) and lysophosphatidyl choline (18:1) are used as early sign object；Acyl group meat poisoning
Alkali C18:0, linoleic acid carnitine, lysophosphatidyl choline (18:3), DL-tryptophan, indole acrylic acid, paracresol sulfate, 3-
Indoxylsulfuric acid salt, cytimidine, kreatinin, sphingosine-1-phosphate, L- Su Shi-dihydrosphingosine and ceramide (d18:0/14:
0) it is used as late-stage markers object；It is evaluated for nephrotic syndrome model lesion and the course of disease.With traditional Biochemical Indices In Serum or kidney
Dirty histopathological analysis is compared, and can be quantified, and is had the characteristics that quick, high sensitivity, high specificity, is able to reflect the lesion of nephrosis
Process.
Detailed description of the invention
Fig. 1 is tissue pathological slice map analysis as a result, in figure: A is C group rat kidney tissue HE dyeing；B is M1 group rat
Renal tissue HE dyeing；C is M2 group rat kidney tissue HE dyeing；D is C group rat kidney tissue Masson dyeing；E is M1 group
Rat kidney tissue Masson dyeing；F is M2 group rat kidney tissue Masson dyeing.
Fig. 2 is to blood serum sample analysis of experimental data figure；In figure: A is C group and M1 group rat blood serum PCA scatter plot；B is C
Group arranges lab diagram with M1 group P of Rats LS-DA；C is C group and M1 group rat blood serum V-S-Plot scatter plot；D is C group and M2 group
Rat blood serum PCA scatter plot；E is that C group and M2 group P of Rats LS-DA arrange lab diagram；F is C group and M2 group rat blood serum V-S-
Plot scatter plot；
Fig. 3 is the relative amount figure and thermal map of progressivity marker；In figure: A is 6 kinds of progressivity marker relative amount figures；B is
The thermal map of 6 kinds of progressivity markers；
Fig. 4 is the correlation analysis figure and correlation analysis figure of 7 kinds of early sign objects and Urine proteins；In figure: A is 7 kinds of early sign objects
With the correlation analysis figure of Urine proteins；B is the ROC curve figure of 7 kinds of early sign objects；
Fig. 5 is the correlation analysis figure and ROC curve figure of 12 kinds of late-stage markers objects and Urine proteins；In figure: A is 12 kinds of late-stage markers objects
With the correlation analysis figure of Urine proteins；B is the ROC curve figure of 12 kinds of phase in advanced stage markers.
Specific embodiment
Material and instrument: 3000 ultra high efficiency liquid phase phase chromatography of Dionex UltiMate and level four bars-electrostatic field orbit trap
High resolution mass spectrum, (Dionex UltiMate 3000 UHPLC-Q Exactive HR-MS, Thermo-Fisher, USA),
Xcalibur work station (Thermo Fi oil er Scientific Inc., Waltham, Ma, USA), TGL-16 high speed platform
Formula refrigerated centrifuge (Hunan Xiang Yi centrifuge Instrument Ltd.)；KYJ-3 nitrogen evaporator special-purpose air source (Beijing Si Poteke
Skill Co., Ltd)；Sartorius BSA124S assay balance (German Sartorius company).
Reagent: HPLC grades of second are cured, methanol and formic acid (Fisher Scientific company)；(hydrochloride for injection is more for adriamycin
It is soft than star, Shenzhen Wan Le medicine company limited liability company, lot number: 1409E3).
Animal: SPF grades of male SD rats, Beijing Vital River Experimental Animals Technology Co., Ltd. provide, animal licensing
Number SCXK (capital) 2013-0014；Animal feeding room keeps temperature (23 ± 1.5) DEG C, relative humidity (45 ± 15) %.
One, the duplication of Adriamycin-induced Nephropathy and tradition are evaluated
1. the duplication of Adriamycin-induced Nephropathy: male SD rat totally 30, adapting to environment one week, collect the urine of 30 24 h of rat
Liquid, and record volume of urine.It is to remove Urine proteins according to primary dcreening operation is carried out or weight is excessively high with the quantity of proteinuria of rat and weight
With too low unqualified rat, by qualified rat be randomly divided into blank control group (C group) and Adriamycin-induced Nephropathy group (M1 group and
M2 group), every group 7, free water is fed in experimentation；
M1 group rat is in the adriamycin of the 1st day 4 mg/kg of tail vein injection, then every 1 week, in the 8th day 2 mg/ of tail vein injection
The adriamycin of kg replicates mRNA IN ADRIAMYCIN NEPHROPATHY early stage model.
M2 group rat replicates mRNA IN ADRIAMYCIN NEPHROPATHY advanced stage mould in the adriamycin of the 1st day 6 mg/kg of disposable tail vein injection
Type.C group gives isodose physiological saline in an identical manner.
2. the tradition evaluation of Adriamycin-induced Nephropathy:
A, histopathologic slide analyzes: C, M1 and M2 group rat were dissected in the 42nd day intraperitoneal injection chloraldurate, took out right
Kidney is put into 10% formalin fixed.
Specific histopathologic slide analysis process: the first step, film-making；Second step, dehydration and embedding；Third step, slice；The
Four steps, dyeing；5th step, transparent and mounting；6th step, light microscopy checking.
B, quantity of proteinuria is analyzed: during model copy, respectively at the 0th day, the 7th day, the 14th day, the 28th day, the
42 day morning, C, M1 and M2 group rat were respectively placed in rat metabolism cage by 9:00, and Yu Ci 9:00 in morning collects urine, and remembers
Volume of urine is recorded, supernatant is taken after 4 DEG C, 3500 rpm centrifugation, 15 min to the urine sample of collection, it is standby to be placed in -80 DEG C of refrigerators
With.
Quantity of proteinuria measurement is carried out using double pungent butyric acid (BCA) methods, measures 24 h urine absorbance of rat in 562 nm,
Establishing criteria curve calculates urinary protein concentrations.By the way that urinary protein concentrations (mg/mL) are calculated 24 multiplied by 24 h urine volume (mL)
H excretion quantity of urinary protein (Upro).
3. the searching of difference metabolin: the data acquisition of blood serum sample: by the C1 group of collection and M1 group and M2 group serum sample
This progress LC-MS analysis.Before analysis, plasma sample thaws in mixture of ice and water, takes 200 μ L and 800 μ L second are added
Nitrile, after fulling shake uniformly, 13,000 rpm is centrifuged 20 min at 4 DEG C, takes 800 μ L of supernatant, is placed under nitrogen and dries up.Add
After entering 200 μ L, 80% acetonitrile-water (v/v), be vortexed 2 min, and 0.22 μm of filter membrane filtration is analyzed to LC-MS.
Using HESI Ionization mode, spray voltage: anode, 3.5kV；Cathode, 2.5kV.320 DEG C of capillary temperature；Add
300 DEG C of hot device temperature；Sheath gas: 35arb, secondary air speed: 10arb；Scan pattern is Full Scan/dd-MS2, acquisition
Range is m/z100-1500, and cation switches acquisition mode；Resolution ratio uses MS Full Scan 35000 FWHM, MS/
MS17500FWHM, NCE 12.5eV, 25eV and 37.5eV.
All original documents, including C1 group and M1 group and M2 group, are all by Xcalibur work station and Compound
Derived from Discover 2.0, an independent peak value meter m/z and retention time have been obtained.All samples are all made of following parameter
It is studied: S/N threshold value: 3, mass range: 100 ~ 1500 Da, quality tolerance: 5 ppm.
The data processing of blood serum sample: data matrix is imported into SIMCA-P13.0(Umetrics, Umea, Sweden) it is soft
In part.Unsupervised pattern-recognition principal component analysis (PCA) is constructed with C group and M1 group and M2 group, can reflect the original of data
State more intuitively shows the whole difference between different samples；Differentiated with C group and M1 group and M2 group building Partial Least Squares and is divided
It analyses (PLS-DA), to arrange the reliability of experiment (permutation test) evaluation model, and is used for subsequent difference metabolin
Identification；V+S-plot(coefficient > 0.7 is constructed with C group and M1 group and M2 group) combine VIP(> 1) find to the two point
Group contributes biggish difference metabolin, further passes throught- test(p< 0.05) the difference metabolin with conspicuousness is determined.
4. the identification of marker
The identification of adriamycin rat model serum biomarkers: according to S-plot and VIP value, selecting has statistically significant difference
Different mass ions (VIP>1.0 and P<0.05) are determined as the difference object marker of identification.The PCA of these mass ions is analyzed
Display blank group and model group can be distinguished obviously, further combined with other statistical analysis methods, according to blood serum metabolic group
The identification method of metabolite is learned, most using databases such as mass spectrometric data and HMBD, Chemspider, KEGG and Lipidmaps
13 and 18 metabolites that there were significant differences are identified respectively in M1 group and M2 group eventually, they may be the blood of nephrotic syndrome
Clear marker.
The marker of progressivity: M1 group is early stage model, and M2 group is advanced stage model, and the difference metabolin both having is recognized
To be progressivity marker.In order to further appreciate that metabolic alterations, thermal map analysis has been carried out based on biomarker.This
Outside, histogram graph representation is used in the detailed variation of the relative amount of these specific metabolites.Finally determining these metabolites may be
The progressivity marker of nephrotic syndrome.
Early stage biomarker: M1 is that early stage model analyzes C group and M1 group according to S-plot and VIP value, is selected
Surely with the mass ions (VIP>1.0 and P<0.05) of statistically-significant difference, it is determined as the early sign object of identification.With above-mentioned
Potential early sign object and rat quantity of proteinuria are that variable by differentiating the data characteristics of every group of data selects suitable phase
Analysis method is closed, the related coefficient of the two is calculated, determines early sign object；According to area under ROC curve, progressivity mark is assessed
The diagnostic accuracy of object, it is generally recognized that AUC < 0.5 is no diagnosis capability, and 0.5 < AUC < 0.7 is that diagnostic accuracy is lower, 0.7 <
AUC<0.9 is that diagnostic accuracy is preferable, and AUC>0.9 is that diagnostic accuracy is best.With the metabolin of AUC > 0.7, finally it is considered
It can be used as the early sign object of reflection nephrotic syndrome.
Advanced stage biomarker: M2 is that advanced stage model analyzes C group and M2 group according to S-plot and VIP value, is selected
Surely with the mass ions (VIP>1.0 and P<0.05) of statistically-significant difference, it is determined as the late-stage markers object of identification.With above-mentioned
Potential late-stage markers object and rat quantity of proteinuria are that variable by differentiating the data characteristics of every group of data selects suitable phase
Analysis method is closed, the related coefficient of the two is calculated, determines late-stage markers object；According to area under ROC curve, progressivity mark is assessed
The diagnostic accuracy of object.With the metabolin of AUC > 0.7, finally it is considered can be used as the late-stage markers object of reflection nephrotic syndrome.
Two, result
1. model tradition evaluation result: by quantity of proteinuria analysis result (see Table 1) it is found that compared with C group, M group rat modeling
Afterwards quantity of proteinuria present at any time stablize ascendant trend, and in the 14th day have significant difference (p< 0.05), show modeling
Success.Wherein M2 group ratio M1 group quantity of proteinuria result is higher, shows that M2 MODEL DAMAGE is serious indirectly, can be used as advanced stage mould
Type.
1 blank group of table and Adriamycin-induced Nephropathy group rat quantity of proteinuria dynamic analysis
" * " represents the * compared with blank control groupp<0.0
By tissue pathological slice map analysis result (Fig. 1) it is found that compared with C group, in M1 group and M2 group renal cells extremely
Severe hydropic degeneration, tubule basophilla becomes and protein cast is common, to severe fibroblast proliferation, interstitial in fibroblast
To severe cell infiltration in inflammatory cell, part Tubular epithelial cell atrophy, lumen becomes larger, and shows model copy success.Wherein
M2 group ratio M1 group Histopathological lesions are more serious, show that M2 MODEL DAMAGE is serious indirectly, can be used as advanced stage model.
2. the searching of difference metabolin: C group and M1 and M2 group rat blood serum metabolin belong to: reference literature and comparison are marked
Quasi- product identify 13 and 18 (see Table 2)s of compound respectively.
The LC-MS attribution data of main compound in 2 rat blood serum of table
TR represents chemical shift
PCA(is carried out to C group and M1 and M2 group blood serum metabolic profiling data and sees Fig. 2A, 2D), PLS-DA arrangement test (see Fig. 2 B,
2E) and V-S-plot(is shown in Fig. 2 C, 2F), it can be seen that both C group and M1 and M2 group are clearly separated in figure (2A, 2D), illustrate mould
Type replicates successfully.Model parameter (M1:R2Y=0.970, Q2=0.893;M2:R2Y=0.977, Q2=0.859), and R2And Q2
Close to 1, illustrates that built disaggregated model is reliable and stable, can be used for the searching of difference metabolin below.Corresponding V+S-plot(figure
2C, 2F) combine VIP(> 1), and pass throught- test screening, finds and contributes biggish metabolin to grouping.
The marker of progressivity: (4- Ethylbenzyl) sorbierites bis- in M1 group and M2 group, C16 dihydrosphingosine, haemolysis
Significant changes occur for phosphatidyl-ethanolamine (22:6), lysophosphatidyl choline (22:5), N- ethanol amine, proline.It is double
(4- Ethylbenzyl) sorbierite, C16 dihydrosphingosine, N- ethanol amine, this 4 metabolins of proline continue in M1 and M2 group
It reduces, and lysophosphatidyl ethanolamine (22:6), lysophosphatidyl choline (22:5) are persistently increased in M1 and M2 group.It is final true
These fixed metabolites may be the progressivity marker of nephrotic syndrome.
The marker of early stage: using above-mentioned 7 early sign objects and rat quantity of proteinuria as variable, by differentiating every group of number
According to data characteristics, the related coefficient (see Fig. 4 A) both calculated, result of study finds Adriamycin-induced Nephropathy quantity of proteinuria
With oleamide, docosahexaenoic acid, 14S- hydroxyl-docosahexaenoic acid, clupanodonic acid, uric acid, haemolysis phosphorus
Phosphatidylcholine (20:5) is positively correlated, and negatively correlated with lysophosphatidyl choline (18:1), related coefficient is all larger than 0.5.Using
ROC curve assesses (see Fig. 4 B) diagnosis capability of early sign object, finally obtains oleamide, two dodecahexaenes
Acid, 14S- hydroxyl-docosahexaenoic acid, clupanodonic acid, uric acid, lysophosphatidyl choline (20:5) and lysophosphatide
The area under the curve of phatidylcholine (18:1) is all larger than 0.9, can be used as early sign object, is used for nephrotic syndrome model early lesion
Evaluation.
The marker in advanced stage: using above-mentioned 12 late-stage markers objects and rat quantity of proteinuria as variable, by differentiating every group
The data characteristics of data, calculates the related coefficient (see Fig. 5 A) of the two, and result of study finds that Adriamycin-induced Nephropathy Urine proteins are fixed
Amount and acylcarnitines C18:0, linoleic acid carnitine, lysophosphatidyl choline (18:3) is positively correlated, with DL-tryptophan, indoles
Acrylic acid, paracresol sulfate, 3- indoxylsulfuric acid salt, cytimidine, kreatinin, sphingosine-1-phosphate, L- Su Shi-dihydro sheath ammonia
Pure and mild ceramide (d18:0/14:0) is negatively correlated, and related coefficient is all larger than 0.5.Using ROC curve to late-stage markers object
Diagnosis capability is assessed (see Fig. 5 B), finally obtains acylcarnitines C18:0, linoleic acid carnitine, lysophosphatidyl choline
(18:3), DL-tryptophan, indole acrylic acid, paracresol sulfate, 3- indoxylsulfuric acid salt, cytimidine, kreatinin, sphingol -1-
The area under the curve of phosphoric acid and ceramide (d18:0/14:0) is all larger than 0.9, can be used as late-stage markers object, comprehensive for nephrosis
Levy the evaluation of model advanced lesions.
The present invention has finally determined the marker of reflection nephrotic syndrome progressive disease.I.e. with bis- (4- Ethylbenzyl) mountains
Pears alcohol, C16 dihydrosphingosine, lysophosphatidyl ethanolamine (22:6), lysophosphatidyl choline (22:5), N- ethanol amine,
Proline is as progressivity marker；Oleamide, docosahexaenoic acid, 14S- hydroxyl-docosahexaenoic acid, 22
Carbon 5 alkene acid, uric acid, lysophosphatidyl choline (20:5) and lysophosphatidyl choline (18:1) are used as early sign object；Acyl group
Carnitine C18:0, linoleic acid carnitine, lysophosphatidyl choline (18:3), DL-tryptophan, indole acrylic acid, paracresol sulfuric acid
Salt, 3- indoxylsulfuric acid salt, cytimidine, kreatinin, sphingosine-1-phosphate, L- Su Shi-dihydrosphingosine and ceramide (d18:
0/14:0) it is used as late-stage markers object；It is evaluated for nephrotic syndrome model lesion and the course of disease.
Claims (7)
1. a kind of nephrotic syndrome lesion process associated metabolic marker, it is characterised in that: the metabolic markers are bis- (4- second
Base benzyl) sorbierite, C16 dihydrosphingosine, lysophosphatidyl ethanolamine (22:6), lysophosphatidyl choline (22:5),
N- ethanol amine, proline is as progressivity marker；Oleamide, docosahexaenoic acid, 12 carbon six of 14S- hydroxyl-two
Olefin(e) acid, clupanodonic acid, uric acid, lysophosphatidyl choline (20:5) and lysophosphatidyl choline (18:1) are as early stage
Marker；Acylcarnitines C18:0, linoleic acid carnitine, lysophosphatidyl choline (18:3), DL-tryptophan, indole acrylic acid,
Paracresol sulfate, 3- indoxylsulfuric acid salt, cytimidine, kreatinin, sphingosine-1-phosphate, L- Su Shi-dihydrosphingosine and mind
Late-stage markers object is used as through amide (d18:0/14:0).
2. screening a kind of method of nephrotic syndrome lesion process associated metabolic marker described in claim 1, feature exists
In: steps are as follows:
The duplication of (1) two kind of Adriamycin-induced Nephropathy and tradition are evaluated: rat is randomly divided into blank control group, early stage adriamycin
Nephropathy model group and advanced stage Adriamycin-induced Nephropathy group, early stage Adriamycin-induced Nephropathy group and advanced stage Adriamycin-induced Nephropathy group are adopted
It is induced with tail vein injection adriamycin, establishes Adriamycin-induced Nephropathy；Utilize renal pathology degree of injury and Urine proteins
Quantitative level evaluates Adriamycin-induced Nephropathy；
(2) searching of difference metabolin: collecting the blood serum sample of each group rat in the different time stage, carries out LC-MS to serum
Analysis, belongs to metabolin out；Liquid matter map is converted to data matrix through processing, imported into SIMCA-PSoftware carries out difference metabolism
Object is found；
(3) marker for combining statistical method screening, determining reflection nephrotic syndrome lesion process；
(4) based on the relative amount of metabolin, metabolin is confirmed using thermal map, correlation analysis figure, and uses ROC curve
The diagnosis capability of marker is assessed, early stage, advanced stage and the progressivity mark of reaction nephrotic syndrome model are finally determined
Will object.
3. the method for screening nephrotic syndrome lesion process associated metabolic marker according to claim 2, feature exist
In: the duplication of two kinds of Adriamycin-induced Nephropathies in step (1) method particularly includes: rat adapts to environment one week, collects rat 24
The urine of h, and record volume of urine；It is that foundation carries out primary dcreening operation with the quantity of proteinuria of rat and weight, qualified rat is random
It is divided into blank control group and early stage Adriamycin-induced Nephropathy group and advanced stage Adriamycin-induced Nephropathy group, free water in experimentation
Feed；Early stage Adriamycin-induced Nephropathy group rat is in the adriamycin of the 1st day 4 mg/kg of tail vein injection, then every 1 week, in the 8th
The adriamycin of 2 mg/kg of its tail vein injection replicates mRNA IN ADRIAMYCIN NEPHROPATHY early stage model；Advanced stage Adriamycin-induced Nephropathy group rat in
The adriamycin of 1st day 6 mg/kg of disposable tail vein injection replicates mRNA IN ADRIAMYCIN NEPHROPATHY advanced stage model；Blank control group is with identical
Mode give isodose physiological saline；
During model copy, respectively at the 0th day, the 7th day, the 14th day, the 28th day, 9:00 in the 42nd day morning by blank control
Group and early and late Adriamycin-induced Nephropathy group rat are respectively placed in rat metabolism cage, and Yu Ci 9:00 in morning collects urine
Liquid, and volume of urine is recorded, supernatant is taken after 4 DEG C, 3500 rpm centrifugation, 15 min to the urine sample of collection, is placed in -80
DEG C refrigerator is spare；
Quantity of proteinuria analysis is carried out using double pungent butyric acid BCA methods to urine, 24 h urine of rat is quantitative determined at 562 nm
Absorbance, establishing criteria curve calculate urine protein concentration, are counted by the way that urinary protein concentrations mg/mL is urinated volume mL multiplied by 24 h
Calculate 24 h excretion quantity of urinary protein Upro.
4. the method for screening nephrotic syndrome lesion process associated metabolic marker according to claim 2, feature exist
In: blood serum sample carries out LC-MS analysis method particularly includes: uses HESI Ionization mode, spray voltage: anode, 3.5kV；It is negative
Pole, 2.5kV；320 DEG C of capillary temperature；300 DEG C of heter temperature；Sheath gas: 35arb, secondary air speed: 10arb；Scanning
Mode is Full Scan/dd-MS2, acquisition range m/z100-1500, cation switching acquisition mode；Resolution ratio uses MS
Full Scan 35000 FWHM, MS/MS17500FWHM, NCE 12.5eV, 25eV and 37.5eV；
All original documents, including C1 group and M1 group and M2 group, are all by Xcalibur work station and Compound
Derived from Discover 2.0, an independent peak value meter m/z and retention time have been obtained；All samples are all made of following parameter
It is studied: S/N threshold value: 3, mass range: 100 ~ 1500 Da, quality tolerance: 5 ppm.
5. the method for screening nephrotic syndrome lesion process associated metabolic marker according to claim 2, feature exist
In: blood serum sample data processing method in step (2) are as follows: data matrix is imported into SIMCA-PIn 13.0 softwares, with blank pair
Unsupervised pattern-recognition principal component analysis PCA is constructed with early and late Adriamycin-induced Nephropathy group according to group；With blank control
Group constructs Partial Least Squares discriminant analysis PLS-DA with early stage Adriamycin-induced Nephropathy group and advanced stage Adriamycin-induced Nephropathy group,
To arrange the reliability of experimental evaluation model, and it is used for the identification of subsequent difference metabolin；Construct V+S-plot
Coefficient > 0.7 combines VIP > 1 to find and contributes biggish difference metabolin to the two grouping, passes throught- test(p< 0.05)
Determine the difference metabolin with conspicuousness.
6. the method for screening nephrotic syndrome lesion process associated metabolic marker according to claim 2, feature exist
In: step (3) method particularly includes: according to S-plot and VIP value, the selected mass ions VIP with statistically-significant difference >
1.0 and P < 0.05, it is determined as the difference object marker of identification；By the difference metabolin that early and late model has be considered into
Malleability marker；
According to S-plot and VIP value, blank control group and early stage Adriamycin-induced Nephropathy group are analyzed, selecting has statistics
Mass ions VIP>1.0 and P<0.05 for learning significant difference, are determined as potential early sign object；With above-mentioned potential early sign object
It is variable with rat quantity of proteinuria, by differentiating the data characteristics of every group of data, selects correlation analysis method, calculate the two
Related coefficient determines early sign object；According to area under ROC curve, using the metabolin of AUC > 0.7 as reflection nephrotic syndrome
Early sign object；
According to S-plot and VIP value, blank control group and advanced stage Adriamycin-induced Nephropathy group are analyzed, selecting has statistics
Mass ions VIP>1.0 and P<0.05 for learning significant difference, are determined as potential late-stage markers object；With above-mentioned potential late-stage markers object
It is variable with rat quantity of proteinuria, by differentiating the data characteristics of every group of data, selects correlation analysis method, calculate the two
Related coefficient determines late-stage markers object；According to area under ROC curve, using the metabolin of AUC > 0.7 as reflection nephrotic syndrome
Late-stage markers object.
7. a kind of application of nephrotic syndrome lesion process associated metabolic marker described in claim 1, it is characterised in that: institute
State purposes of the metabolic markers in preparation nephrotic syndrome lesion process diagnosis different reagent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111413431A (en) * | 2020-04-17 | 2020-07-14 | 中国药科大学 | Method for screening metabolic markers of drug-induced acute kidney injury lesion process |
| CN111426766A (en) * | 2020-04-17 | 2020-07-17 | 中国药科大学 | Construction and evaluation method of drug-induced acute kidney injury mouse model |
| CN111896728A (en) * | 2020-07-07 | 2020-11-06 | 山西大学 | Combined marker for detecting anti-depression amount-effect and amount-toxicity relationship of bupleurum petroleum ether part and application thereof |
| CN114026427A (en) * | 2020-05-19 | 2022-02-08 | 深圳市中医院 | Marker for diagnosing renal disease and diagnostic method |
| CN116068161A (en) * | 2023-03-07 | 2023-05-05 | 中国中医科学院中药研究所 | Biomarker for diagnosing liver diseases and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901789A (en) * | 2012-09-21 | 2013-01-30 | 中国药科大学 | Determination method of serum metabolic marker for early diagnosis of diabetic nephropathy. |
| WO2016115496A1 (en) * | 2015-01-15 | 2016-07-21 | Joslin Diabetes Center | Metabolite biomarkers predictive of renal disease in diabetic patients |
| CN108918571A (en) * | 2018-06-15 | 2018-11-30 | 山西大学 | Application of the metabolic markers in preparation nephrotic syndrome lesion process diagnosis different reagent |
| CN109507337A (en) * | 2018-12-29 | 2019-03-22 | 上海交通大学医学院附属新华医院 | A kind of new method based on blood urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism |
-
2019
- 2019-06-13 CN CN201910509827.8A patent/CN110286189B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901789A (en) * | 2012-09-21 | 2013-01-30 | 中国药科大学 | Determination method of serum metabolic marker for early diagnosis of diabetic nephropathy. |
| WO2016115496A1 (en) * | 2015-01-15 | 2016-07-21 | Joslin Diabetes Center | Metabolite biomarkers predictive of renal disease in diabetic patients |
| CN108918571A (en) * | 2018-06-15 | 2018-11-30 | 山西大学 | Application of the metabolic markers in preparation nephrotic syndrome lesion process diagnosis different reagent |
| CN109507337A (en) * | 2018-12-29 | 2019-03-22 | 上海交通大学医学院附属新华医院 | A kind of new method based on blood urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism |
Non-Patent Citations (3)
| Title |
|---|
| LI LYU 等: "Serum Metabolic Profiling in a Mouse Model of Adriamycin-Induced Focal Segmental Glomerulosclerosis", 《CHINESE MEDICAL JOURNAL》 * |
| 张王宁 等: "基于代谢组学的阿霉素肾病大鼠模型损伤程度评价", 《中草药》 * |
| 皮子凤等: "五味子治疗大鼠糖尿病肾病作用机制的血清代谢组学研究 ", 《分析化学》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111413431A (en) * | 2020-04-17 | 2020-07-14 | 中国药科大学 | Method for screening metabolic markers of drug-induced acute kidney injury lesion process |
| CN111426766A (en) * | 2020-04-17 | 2020-07-17 | 中国药科大学 | Construction and evaluation method of drug-induced acute kidney injury mouse model |
| CN111413431B (en) * | 2020-04-17 | 2022-03-01 | 中国药科大学 | Method for screening metabolic markers of drug-induced acute kidney injury lesion process |
| CN114026427A (en) * | 2020-05-19 | 2022-02-08 | 深圳市中医院 | Marker for diagnosing renal disease and diagnostic method |
| CN114026427B (en) * | 2020-05-19 | 2023-11-17 | 深圳市中医院 | Marker for diagnosing kidney disease and diagnostic method |
| CN111896728A (en) * | 2020-07-07 | 2020-11-06 | 山西大学 | Combined marker for detecting anti-depression amount-effect and amount-toxicity relationship of bupleurum petroleum ether part and application thereof |
| CN111896728B (en) * | 2020-07-07 | 2023-10-13 | 山西大学 | Combined marker for detecting antidepressant quantity-effect and quantity-toxicity relation of petroleum ether part of bupleurum and application thereof |
| CN116068161A (en) * | 2023-03-07 | 2023-05-05 | 中国中医科学院中药研究所 | Biomarker for diagnosing liver diseases and application thereof |
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